A versatile approach for site-directed spin labeling and structural EPR studies of RNAsElectronic supplementary information (ESI) available: Supplementary Fig. S1-S2, original PELDOR/DEER time traces (Fig. S3) of data shown in Fig. 2, and supplementary CW EPR studies (Fig. S4). See DOI: 10.1039/c3ob42154f

• Main Authors: Babaylova, Elena S, Ivanov, Anton V, Malygin, Alexey A, Vorobjeva, Maria A, Venyaminova, Alia G, Polienko, Yuliya F, Kirilyuk, Igor A, Krumkacheva, Olesya A, Fedin, Matvey V, Karpova, Galina G, Bagryanskaya, Elena G
• Format: Article
• Language: English
• ISSN: 1477-0520; 1477-0539
• DOI: 10.1039/c3ob42154f

Site-directed spin labeling (SDSL) is widely applied for structural studies of biopolymers by electron paramagnetic resonance (EPR). However, SDSL of long RNA sequences still remains a challenging task. Here, we propose a novel SDSL approach potentially suitable for long natural RNAs, which is based on the attachment of a linker containing an aliphatic amino group to the target nucleotide residue followed by selective coupling of a spin label to this amino group. Such a linker can be attached to the desired RNA residue via a sequence-specific reaction with the derivatives of oligodeoxyribonucleotides. To verify this approach, we applied it to model RNA duplex with known structure and expected distance between corresponding residues. A new 2,5-bis(spirocyclohexane)-substituted spin label with advanced stability and relaxation properties has been used, and the distance distribution measured using Q-band (34 GHz) pulsed double electron-electron resonance corresponds well to the expected one. We have additionally validated the obtained results by studying a similar RNA duplex, where the linker with the aliphatic amino group was introduced via solid-phase synthesis. Although this novel SDSL approach does not provide an advantage in precision of molecular distance measurements, we believe that its applicability to long RNAs is a crucial benefit for future structural studies using pulse EPR. We propose and validate a new site-directed spin labeling approach affording EPR distance measurements in long RNAs.